In general, the invention relates to methods and compounds used to decrease necrosis.
In many diseases, cell death is mediated through apoptotic and/or necrotic pathways. While much is known about the mechanisms of action that control apoptosis, control of necrosis is not as well understood. Understanding the mechanisms regulating both necrosis and apoptosis in cells is essential to being able to treat conditions, such as neurodegenerative diseases, stroke, coronary heart disease, kidney disease, and liver disease. A thorough understanding of necrotic and apoptotic cell death pathways is also crucial to treating AIDS and the conditions associated with AIDS, such as retinal necrosis.
Research has shown that caspases play a central role in the induction of apoptosis. Peptide based inhibitors of caspases, such as zVAD-fmk are useful in preventing activation of the apoptotic cell death pathway in cells stimulated to undergo apoptosis by compounds such as TNFxcex1. However, cells treated with zVAD-fmk and these cell death stimuli still die through a caspase-independent form of necrosis.
Discovery of a compound which prevents caspase-independent cell death (necrosis) would provide a useful therapeutic for treating conditions in which necrosis occurs, and for preventing the onset of necrosis. These compounds and methods may be particularly useful for treating ischemic brain and heart injuries and head traumas.
The present invention features methods and compounds for decreasing necrosis. The compounds of the present invention may be used as therapeutics to decrease necrosis in a desired cell, such as a neuron. These compounds are characterized by their ability to decrease necrosis in response to modulation of intracellular signaling pathways, such as those activated by TNFxcex1. By also treating the cells with zVAD-fmk, we have inhibited the apoptotic pathway. Accordingly, we have been able to determine that the compounds of the invention specifically decrease necrosis. In addition, we have shown that the identified compounds that decrease necrosis in response to a necrotic pathway activated by zVAD-fmk and TNFxcex1, also decrease necrosis in response to a necrotic pathway activated by zVAD-fmk and dimethyl sulfoxide (DMSO).
Accordingly, in a first aspect, the invention features a chemical compound in a pharmaceutically acceptable carrier, having the formula: 
wherein each R1 is independently selected from the group consisting of hydrogen, carboxy, methyl, hydroxyl, methoxyl, amino, and nitro; R2 is selected from the group consisting of hydrogen, alkyl, and acyl; R3 is selected from the group consisting of alkyl, acyl, halogen, hydrogen, or hydroxyl; R4 is selected from the group consisting of methyl, hydroxyl, carboxyl, and linear and branching alkyl groups; X is selected from the group consisting of xe2x95x90O, xe2x80x94OH and xe2x80x94H; Y is selected from the group consisting of xe2x95x90S and xe2x80x94SR5, where R5 is either hydrogen or an alkyl group; and each of the bonds (a), (b), and (c) independently is either a double or single bond, provided, however, that bond (a) and bond (b) are not both double bonds.
In a preferred embodiment of the first aspect of the invention, in the compound each R1 is hydrogen; R2 and R3 are each hydrogen; R4 is a methyl group; X is xe2x95x90O; Y is xe2x95x90S; bond (a) is a double bond; and bonds (b) and (c) are each single bonds.
In another embodiment, the acyl group of R1 or R3 is selected from the group consisting of: 
In other embodiments, in the compound if R1 is a hydrogen, then R2 and R3 are not each hydrogen; or R4 is not a methyl group; or X is not xe2x95x90O; or Y is not xe2x95x90S; or bond (a) is not a double bond; or bonds (b) and (c) are not each single bonds. If R2 is a hydrogen, then R1 is a not a hydrogen, or R3 is not a hydrogen; or R4 is not a methyl group; or X is not xe2x95x90O; or Y is not xe2x95x90S; or bond (a) is not a double bond; or bonds (b) and (c) are not each single bonds. If R3 is a hydrogen, then R1 is a not a hydrogen, or R2 is not a hydrogen; or R4 is not a methyl group; or X is not xe2x95x90O; or Y is not xe2x95x90S; or bond (a) is not a double bond; or bonds (b) and (c) are not each single bonds. If R4 is a methyl group, then R1 is a not a hydrogen, or R2 and R3 are not each not a hydrogen; or X is not xe2x95x90O; or Y is not xe2x95x90S; or bond (a) is not a double bond; or bonds (b) and (c) are not each single bonds.
In other embodiments, if X is xe2x95x90O, then R1 is a not a hydrogen, or R2 and R3 are not each not a hydrogen; or R4 is not a methyl group; or Y is not xe2x95x90S; or bond (a) is not a double bond; or bonds (b) and (c) are not each single bonds. If Y is xe2x95x90S, then R1 is a not a hydrogen, or R2 and R3 are not each not a hydrogen; or R4 is not a methyl group; or X is not xe2x95x90O; or bond (a) is not a double bond; or bonds (b) and (c) are not each single bonds.
In yet other embodiments, if bond (a) is a double bond, then R1 is a not a hydrogen, or R2 and R3 are not each not a hydrogen; or R4 is not a methyl group; or X is not xe2x95x90O; or Y is not xe2x95x90S; or bonds (b) and (c) are not each single bonds. If bond (b) is a single bond, then R1 is a not a hydrogen, or R2 and R3 are not each not a hydrogen; or R4 is not a methyl group; or X is not xe2x95x90O; or Y is not xe2x95x90S; bond (a) is not a double bond or bond (c) is not a single bond. If bond (c) is a single bond, then R1 is a not a hydrogen, or R2 and R3 are not each not a hydrogen; or R4 is not a methyl group; or X is not xe2x95x90O; or Y is not xe2x95x90S; bond (a) is not a double bond or bond (b) is not a single bond.
In a second aspect, the invention features a compound in a pharmaceutically acceptable carrier, having the formula: 
wherein each of X1 and X2 is independently selected from the group consisting of xe2x95x90O, xe2x80x94OH and xe2x80x94H; R1 is selected from the group consisting of hydrogen and hydroxyl; R2 is selected from the group consisting of hydrogen, sulfate, nitro, and halide; and the bond (a) is either a single or double bond.
In a preferred embodiment of the second aspect of the invention, in the compound each of X1 and X2 is xe2x95x90O; R1 is a hydroxyl group; R2 is a nitro group; and the bond (a) is a double bond.
In other embodiments, if X1 is xe2x95x90O, then X2 is not xe2x95x90O; or R1 is not a hydroxyl group; or R2 is a not a nitro group; or the bond (a) is not a double bond. If X2 is xe2x95x90O, then X1 is not xe2x95x90; or R1 is not a hydroxyl group; or R2 is not a nitro group; or the bond (a) is not a double bond. If R1 is a hydroxyl group, then each of X1 and X2 are not xe2x95x90O; or R2 is a not a nitro group; or the bond (a) is not a double bond. If R2 is a nitro group, then each of X1 and X2 are not xe2x95x90O; or R1 is not a hydroxyl group; or the bond (a) is not a double bond. If the bond (a) is a double bond, then each of X1 and X2 are not xe2x95x90O; or R1 is not a hydroxyl group; or R2 is a not a nitro group; or the bond (a) is not a double bond.
In a third aspect, the invention features a chemical compound in a pharmaceutically acceptable carrier, having the formula: 
wherein each R1 and R2 is independently selected from the group consisting of hydrogen, amino, halide, and hydroxyl; R3 is selected from the group consisting of hydrogen and methyl; and the bond (a) is either a single or double bond.
In a preferred embodiment of the third aspect of the invention, in the compound each R1 is hydrogen; R2 is fluorine; R3 is a methyl group; and the bond (a) is a double bond.
In other embodiments of the third aspect of the invention, if R1 is a hydrogen, then R2 is not fluorine; or R3 is not a methyl group; or the bond (a) is not a double bond. If R2 is a fluorine, then R1 is not hydrogen; or R3 is a not a methyl group; or the bond (a) is not a double bond. If R3 is a methyl group, then R1 is not hydrogen, or R2 is not fluorine; or the bond (a) is not a double bond. If the bond (a) is a double bond, then R1 is not hydrogen, or R2 is not fluorine; or R3 is not a methyl group.
In a fourth aspect, the invention features a chemical compound in a pharmaceutically acceptable carrier, having the formula: 
wherein each R is independently selected from the group consisting of H or CH3; the bond (a) is either a single or double bond; the bond (b) is either a single or double bond; and X is selected from the group consisting of xe2x95x90O, xe2x80x94OH and xe2x80x94H.
In a preferred embodiment of the fourth aspect of the invention, in the compound each R is CH3; the bonds (a) and (b) are each a double bond; and X is xe2x95x90O.
In other embodiments of the fourth aspect of the invention, if each R is CH3, then the bonds (a) and (b) are not each a double bond; or X is not xe2x95x90O. If the double bond (a) is a double bond, then each R is not CH3; or the bond (b) is not a double bond; or X is not xe2x95x90O. If the bond (b) is a double bond, then each R is not CH3; or the bond (a) is not a double bond; or X is not xe2x95x90O. If X is xe2x95x90O, then R is not CH3, or the bonds (a) and (b) are not each a double bond.
In a fifth aspect, the invention features a method for decreasing necrosis, involving contacting a cell with a chemical compound having the formula: 
wherein each R1 is independently selected from the group consisting of hydrogen, carboxy, methyl, hydroxyl, methoxyl, amino, and nitro; R2 is selected from the group consisting of hydrogen, alkyl, and acyl; R3 is selected from the group consisting of alkyl, acyl, halogen, hydrogen, or hydroxyl; R4 is selected from the group consisting of methyl, hydroxyl, carboxyl, and linear and branching alkyl groups; X is selected from the group consisting of xe2x95x90O, xe2x80x94OH and xe2x80x94H; Y is selected from the group consisting of xe2x95x90S and xe2x80x94SR5, where R5 is either hydrogen or an alkyl group; and each of the bonds (a), (b), and (c) independently is either a double or single bond, provided, however, that bond (a) and bond (b) are not both double bonds.
In a preferred embodiment of the fifth aspect of the invention, in the compound each R1 is hydrogen; R2 and R3 are each hydrogen; R4 is a methyl group; X is xe2x95x90O; Y is xe2x95x90S; bond (a) is a double bond; and bonds (b) and (c) are each single bonds.
In another embodiment, the acyl group of R1 or R3 is selected from the group consisting of: 
In other embodiments, in the compound if R1 is a hydrogen, then R2 and R3 are not each hydrogen; or R is not a methyl group; or X is not xe2x95x90O; or Y is not xe2x95x90S; or bond (a) is not a double bond; or bonds (b) and (c) are not each single bonds. If R2 is a hydrogen, then R1 is a not a hydrogen, or R3 is not a hydrogen; or R4 is not a methyl group; or X is not xe2x95x90O; or Y is not xe2x95x90S; or bond (a) is not a double bond; or bonds (b) and (c) are not each single bonds. If R3 is a hydrogen, then R1 is a not a hydrogen, or R2 is not a hydrogen; or R4 is not a methyl group; or X is not xe2x95x90O; or Y is not xe2x95x90S; or bond (a) is not a double bond; or bonds (b) and (c) are not each single bonds. If R4 is a methyl group, then R1 is a not a hydrogen, or R2 and R3 are not each not a hydrogen; or X is not xe2x95x90O; or Y is not xe2x95x90S; or bond (a) is not a double bond; or bonds (b) and (c) are not each single bonds.
In other embodiments, if X is xe2x95x90O, then R1 is a not a hydrogen, or R2 and R3 are not each not a hydrogen; or R4 is not a methyl group; or Y is not xe2x95x90S; or bond (a) is not a double bond; or bonds (b) and (c) are not each single bonds. If Y is xe2x95x90S, then R1 is a not a hydrogen, or R2 and R3 are not each not a hydrogen; or R4 is not a methyl group; or X is not xe2x95x90O; or bond (a) is not a double bond; or bonds (b) and (c) are not each single bonds.
In yet other embodiments, if bond (a) is a double bond, then R1 is a not a hydrogen, or R2 and R3 are not each not a hydrogen; or R4 is not a methyl group; or X is not xe2x95x90O; or Y is not xe2x95x90S; or bonds (b) and (c) are not each single bonds. If bond (b) is a single bond, then R1 is a not a hydrogen, or R2 and R3 are not each not a hydrogen; or R4 is not a methyl group; or X is not xe2x95x90O; or Y is not xe2x95x90S; bond (a) is not a double bond or bond (c) is not a single bond. If bond (c) is a single bond, then R1 is a not a hydrogen, or R2 and R3 are not each not a hydrogen; or R4 is not a methyl group; or X is not xe2x95x90O; or Y is not xe2x95x90S; bond (a) is not a double bond or bond (b) is not a single bond.
In a sixth aspect, the invention features a method for decreasing necrosis, involving contacting a cell with a chemical compound having the formula: 
wherein each of X1 and X2 is independently selected from the group consisting of xe2x95x90O, xe2x80x94OH and xe2x80x94H; R1 is selected from the group consisting of hydrogen and a hydroxyl; R2 is selected from the group consisting of hydrogen, sulfate, nitro, and halide; and the bond (a) is either a single or double bond.
In a preferred embodiment of the sixth aspect of the invention, in the compound each of X1 and X2 is xe2x95x90O; R1 is a hydroxyl group; R2 is a nitro group; and the bond (a) is a double bond.
In other preferred embodiments of the sixth aspect of the invention, if X1 is xe2x95x90O, then X2 is not xe2x95x90O; or R1 is not a hydroxyl group; or R2 is a not a nitro group; or the bond (a) is not a double bond. If X2 is xe2x95x90O, then X1 is not xe2x95x90O; or R1 is not a hydroxyl group; or R2 is not a nitro group; or the bond (a) is not a double bond. If R1 is a hydroxyl group, then each of X1 and X2 are not xe2x95x90O; or R2 is a not a nitro group; or the bond (a) is not a double bond. If R2 is a nitro group, then each of X1 and X2 are not xe2x95x90O; or R1 is not a hydroxyl group; or the bond (a) is not a double bond. If the bond (a) is a double bond, then each of X1 and X2 are not xe2x95x90O; or R1 is not a hydroxyl group; or R2 is a not a nitro group; or the bond (a) is not a double bond.
In a seventh aspect, the invention features a method for decreasing necrosis, involving contacting a cell with a chemical compound having the formula: 
wherein each R1 and R2 is independently selected from the group consisting of hydrogen, amino, halide, and hydroxyl; R3 is selected from the group consisting of hydrogen and methyl; and the bond (a) is either a single or double bond.
In a preferred embodiment of the seventh aspect of the invention, in the compound each R1 is hydrogen; R2 is fluorine; R3 is a methyl group; and the bond (a) is a double bond.
In other embodiments of the seventh aspect of the invention, if R1 is a hydrogen, then R2 is not fluorine; or R3 is not a methyl group; or the bond (a) is not a double bond. If R2 is a fluorine, then R1 is not hydrogen; or R3 is a not a methyl group; or the bond (a) is not a double bond. If R3 is a methyl group, then R1 is not hydrogen, or R2 is not fluorine; or the bond (a) is not a double bond. If the bond (a) is a double bond, then R1 is not hydrogen, or R2 is not fluorine; or R3 is not a methyl group.
In an eighth aspect, the invention features a method for decreasing necrosis, involving contacting a cell with a chemical compound having the formula: 
wherein each R is independently selected from the group consisting of H or CH3; the bond (a) is either a single or double bond; the bond (b) is either a single or double bond; and X is selected from the group consisting of xe2x95x90O, xe2x80x94OH and xe2x80x94H.
In a preferred embodiment of the eighth aspect of the invention, in the compound each R is CH3; the (a) and (b) bonds are each a double bond; and X is xe2x95x90O.
In other embodiments of the eighth aspect of the invention, if each R is CH3, then the bonds (a) and (b) are not each a double bond; or X is not xe2x95x90O. If the double bond (a) is a double bond, then each R is not CH3; or the bond (b) is not a double bond; or X is not xe2x95x90O. If the bond (b) is a double bond, then each R is not CH3; or the bond (a) is not a double bond; or X is not xe2x95x90O. If X is xe2x95x90O, then R is not CH3, or the bonds (a) and (b) are not each a double bond.
In a preferred embodiment of any of the fifth, sixth, seventh, or eighth aspects of the invention, the cell is capable of undergoing necrosis in the presence of zVAD-fmk and TNFxcex1. In another preferred embodiment, the cell is capable of undergoing necrosis in the presence of zVAD-fmk and DMSO. In yet another preferred embodiment, the cell is mammalian, such as a human or rodent cell. In yet another preferred embodiment, the cell is a neuron. In still another preferred embodiment, the compound is in a pharmaceutically acceptable carrier.
In a ninth aspect, the invention features a method for treating a condition in a subject, involving the steps of administering a chemical compound having the formula: 
to the subject, in a dosage sufficient to decrease necrosis, wherein each R1 is independently selected from the group consisting of hydrogen, carboxy, methyl, hydroxyl, methoxyl, amino, and nitro; R2 is selected from the group consisting of hydrogen, alkyl, and acyl; R3 is selected from the group consisting of alkyl, acyl, halogen, hydrogen, or hydroxyl; R4 is selected from the group consisting of methyl, hydroxyl, carboxyl, and linear and branching alkyl groups; X is selected from the group consisting of xe2x95x90O, xe2x80x94OH and xe2x80x94H; Y is selected from the group consisting ofxe2x95x90S and xe2x80x94SR5, where R is either hydrogen or an alkyl group; and each of the bonds (a), (b), and (c) independently is either a double or single bond, provided, however, that bond (a) and bond (b) are not both double bonds.
In a preferred embodiment of the ninth aspect of the invention, in the compound each R1 is hydrogen; R2 and R3 are each hydrogen; R4 is a methyl group; X is xe2x95x90O; Y is xe2x95x90S; bond (a) is a double bond; and bonds (b) and (c) are each single bonds.
In another embodiment, the acyl group of R1 or R3 is selected from the group consisting of: 
In other embodiments, in the compound if R1 is a hydrogen, then R2 and R3 are not each hydrogen; or R4 is not a methyl group; or X is not xe2x95x90O; or Y is not xe2x95x90S; or bond (a) is not a double bond; or bonds (b) and (c) are not each single bonds. If R2 is a hydrogen, then R1 is a not a hydrogen, or R3 is not a hydrogen; or R4 is not a methyl group; or X is not xe2x95x90O; or Y is not xe2x95x90S; or bond (a) is not a double bond; or bonds (b) and (c) are not each single bonds. If R3 is a hydrogen, then R1 is a not a hydrogen, or R2 is not a hydrogen; or R4 is not a methyl group; or X is not xe2x95x90O; or Y is not xe2x95x90S; or bond (a) is not a double bond; or bonds (b) and (c) are not each single bonds. If R4 is a methyl group, then R1 is a not a hydrogen, or R2 and R3 are not each not a hydrogen; or X is not xe2x95x90O; or Y is not xe2x95x90S; or bond (a) is not a double bond; or bonds (b) and (c) are not each single bonds.
In other embodiments, if X is xe2x95x90O, then R1 is a not a hydrogen, or R2 and R3 are not each not a hydrogen; or R4 is not a methyl group; or Y is not xe2x95x90S; or bond (a) is not a double bond; or bonds (b) and (c) are not each single bonds. If Y is xe2x95x90S, then R1 is a not a hydrogen, or R2 and R3 are not each not a hydrogen; or R4 is not a methyl group; or X is not xe2x95x90O; or bond (a) is not a double bond; or bonds (b) and (c) are not each single bonds.
In yet other embodiments, if bond (a) is a double bond, then R1 is a not a hydrogen, or R2 and R3 are not each not a hydrogen; or R4 is not a methyl group; or X is not xe2x95x90O; or Y is not xe2x95x90S; or bonds (b) and (c) are not each single bonds. If bond (b) is a single bond, then R1 is a not a hydrogen, or R2 and R3 are not each not a hydrogen; or R4 is not a methyl group; or X is not xe2x95x90O; or Y is not xe2x95x90S; bond (a) is not a double bond or bond (c) is not a single bond. If bond (c) is a single bond, then R1 is a not a hydrogen, or R2 and R3 are not each not a hydrogen; or R4 is not a methyl group; or X is not xe2x95x90O; or Y is not xe2x95x90S; bond (a) is not a double bond or bond (b) is not a single bond.
In a tenth aspect, the invention features a method for treating a condition in a subject, involving the steps of administering a chemical compound having the formula: 
to the subject, in a dosage sufficient to decrease necrosis, wherein each of X1 and X2 is independently selected from the group consisting of xe2x95x90O, xe2x80x94OH and xe2x80x94H; R1 is selected from the group consisting of hydrogen and a hydroxyl; R2 is selected from the group consisting of hydrogen, sulfate, nitro, and halide; and the bond (a) is either a single or double bond.
In a preferred embodiment of the tenth aspect of the invention, in the compound each of X1 and X2 is xe2x95x90O; R1 is a hydroxyl group; R2 is a nitro group; and the bond (a) is a double bond.
In other embodiments of the tenth aspect of the invention, if X1 is xe2x95x90O, then X2 is not xe2x80x94O; or R1 is not a hydroxyl group; or R2 is a not a nitro group; or the bond (a) is not a double bond. If X2 is xe2x95x90O, then X1 is not xe2x95x90O; or R1 is not a hydroxyl group; or R2 is not a nitro group; or the bond (a) is not a double bond. If R1 is a hydroxyl group, then each of X1 and X2 are not xe2x95x90O; or R2 is a not a nitro group; or the bond (a) is not a double bond. If R2 is a nitro group, then each of X1 and X2 are not xe2x95x90O; or R1 is not a hydroxyl group; or the bond (a) is not a double bond. If the bond (a) is a double bond, then each of X1 and X2 are not xe2x95x90O; or R1 is not a hydroxyl group; or R2 is a not a nitro group; or the bond (a) is not a double bond.
In an eleventh aspect, the invention features a method for treating a condition in a subject, involving the steps of administering a chemical compound having the formula: 
to the subject, in a dosage sufficient to decrease necrosis, wherein each R1 and R2 is independently selected from the group consisting of hydrogen, amino, halide, and hydroxyl; R3 is selected from the group consisting of hydrogen and methyl; and the bond (a) is either a single or double bond.
In a preferred embodiment of the eleventh aspect of the invention, in the compound each R1 is hydrogen; R2 is fluorine; R3 is a methyl group; and the bond (a) is a double bond.
In other embodiments of the eleventh aspect of the invention, if R1 is a hydrogen, then R1 is not fluorine; or R3 is not a methyl group; or the bond (a) is not a double bond. If R2 is a fluorine, then R1 is not hydrogen; or R3 is a not a methyl group; or the bond (a) is not a double bond. If R3 is a methyl group, then R1 is not hydrogen, or R2 is not fluorine; or the bond (a) is not a double bond. If the bond (a) is a double bond, then R1 is not hydrogen, or R2 is not fluorine; or R3 is not a methyl group.
In a twelfth aspect, the invention features a method for treating a condition in a subject, involving the steps of administering a chemical compound having the formula: 
to the subject, in a dosage sufficient to decrease necrosis, wherein each R is independently selected from the group consisting of H or CH3; the bond (a) is either a single or double bond; the bond (b) is either a single or double bond; and X is selected from the group consisting of xe2x95x90O, xe2x80x94OH and xe2x80x94H.
In a preferred embodiment of the twelfth aspect of the invention, in the compound each R is CH3; the (a) and (b) bonds are each a double bond; and X is xe2x95x90O.
In other embodiments of the twelfth aspect of the invention, if each R is CH3, then the bonds (a) and (b) are not each a double bond; or X is not xe2x95x90O. If the double bond (a) is a double bond, then each R is not CH3; or the bond (b) is not a double bond; or X is not xe2x95x90O. If the bond (b) is a double bond, then each R is not CH3; or the bond (a) is not a double bond; or X is not xe2x95x90O. If X is xe2x95x90O, then R is not CH3, or the bonds (a) and (b) are not each a double bond.
In a preferred embodiment of any of the ninth, tenth, eleventh, or twelfth aspects of the invention, the condition is a neurodegenerative disease. Most preferably the neurodegenerative disease is selected from the group consisting of Alzheimer""s disease, Huntington""s disease, cerebral ischemia, stroke, amyotropic lateral sclerosis, multiple sclerosis, Lewy body disease, Menkes, disease, Wilson disease, Creutzfeldt-Jakob disease, and Fahr disease. In other preferred embodiments, the condition is ischemic brain or heart injury, or head trauma. In another preferred embodiment, the subject is a mammal, such as a human or a rodent.
In a thirteenth aspect, the invention features a method for identifying a compound that decreases necrosis, involving the steps of: providing a cell in which apoptosis is prevented; contacting the cell with a first compound that causes a cell to undergo necrosis; contacting the cell with a second compound; and measuring necrosis relative to a control cell, wherein a decrease in necrosis indicates that the second compound decreases necrosis.
In a preferred embodiment of the thirteenth aspect of the invention, apoptosis is prevented by contacting the cell with zVAD-fmk. In another preferred embodiment, the first compound is TNFxcex1 or DMSO.
It will be appreciated that any of the R, X, or Y groups of the compounds of the invention, or of the compounds used in any method of the invention may be alkyl derivatives or contain alkyl linkers.
As used herein, by xe2x80x9cdecreasing necrosisxe2x80x9d is meant reducing the number of cells which undergo necrosis relative to a control cell, receiving a cell death stimulus, such as TNFxcex1/zVAD-fmk or DMSO/zVAD-fmk without a candidate small molecule inhibitor. Preferably necrosis is decreased 10% relative to a control. More preferably necrosis is decreased 50% relative to a control. Most preferably necrosis is decreased 90% relative to a control. Preferably a decrease in necrosis is tested by determining the ATP level in a cell which has received a test compound, such as a compound from a chemical library, and comparing it to the ATP level in co a control cell. Necrosis is decreased in a cell treated with a test compound in which the ATP level does not decrease as much as it does in the control cell.
By xe2x80x9ctest compoundxe2x80x9d is meant a chemical, be it naturally-occurring or artificially-derived, that is surveyed for its ability to modulate the level of necrosis by employing one of the assay methods described herein. Test compounds may include, for example, peptides, polypeptides, synthesized organic molecules, naturally occurring organic molecules, nucleic acid molecules, and components thereof.
By xe2x80x9ccell deathxe2x80x9d is meant the death of a cell by either apoptosis or necrosis.
As used herein, by xe2x80x9cnecrosisxe2x80x9d is meant caspase-independent cell death characterized by cellular ATP depletion. Preferably the cell is depleted of ATP 10% relative to a control cell, receiving vehicle only (for example, DMSO). More preferably, the cell is depleted of ATP 50% relative to a control cell. Most preferably, the cell is depleted of ATP 90% relative to a control cell. Preferably, necrosis is tested by determining the ATP level in a cell which has received a compound, for example, zVAD-fmk, DMSO, or TNFxcex1, and comparing it to the ATP level in a cell receiving vehicle only. Necrosis occurs in a cell treated with a test compound in which the ATP level decreases relative to the control cell.
Necrosis may be liquifactive, may affect adipose or hepatic tissue, and may be caseous or fibrinoid. A cell may undergo necrosis in response to ischemic cell injury or viral infection.
By xe2x80x9ccaspase-independent cell deathxe2x80x9d is meant cell death that occurs when apoptosis is prevented. Apoptosis may be prevented by contacting a cell with a caspase inhibitor such as zVAD-fmk at a concentration sufficient enough that the cell survives when stimulated to undergo apoptosis, for example, by treatment with an apoptosis-promoting drug or ionizing radiation.
By xe2x80x9capoptosisxe2x80x9d is meant cell death characterized by any of the following properties: nuclear condensation, DNA fragmentation, membrane blebbing, or cell shrinkage.
By xe2x80x9cmodulation of intracellular signaling pathways mediated by TNFxcex1xe2x80x9d is meant a change in the communication between components of a cell in response to contacting the cell with TNFxcex1. The change may be in the way or duration in which proteins within the cell interact, or the way or duration in which proteins are altered, such as by phosphorylation or dephosphorylation, or in the way or duration in which proteins interact with DNA.
By xe2x80x9cmodulation of intracellular signaling pathways mediated by DMSOxe2x80x9d is meant a change in the communication between components of a cell in response to contacting the cell with DMSO. The change may be in the way or duration in which proteins within the cell interact, or the way or duration in which proteins are altered, such as by phosphorylation or dephosphorylation, or in the way or duration in which proteins interact with DNA.
By xe2x80x9ctreatingxe2x80x9d is meant to submit or subject an animal, cell, lysate or extract derived from a cell, or a molecule derived from a cell to a test compound that decreases necrosis.
By xe2x80x9cconditionxe2x80x9d is meant a state of being or feeling. Conditions include, but are not limited to, neurodegenerative disease, stroke, liver disease, pancreatic disease, ischemic brain or heart injury or other ischemic injuries, head trauma, a necrotic ulceration, septic shock, coronary heart disease, gastrointestinal disease, tuberculosis, alteration of blood vessels, viral infection (e.g., HIV infection or AIDS), or conditions associated with HIV infection or AIDS.
By xe2x80x9cneurodegenerative diseasexe2x80x9d is meant a disease characterized by neuronal cell death. Examples of neurodegenerative diseases include, but are not limited to, Alzheimer""s disease, Huntington""s disease and related polyglutamine expansion diseases, cerebral ischemia, stroke, amyotropic lateral sclerosis, multiple sclerosis, Lewy body disease, Menkes disease, Wilson disease, Creutzfeldt-Jakob disease, and Fahr disease.
By xe2x80x9cneuronxe2x80x9d is meant a cell of ectodermal embryonic origin derived from any part of the nervous system of an animal. Neurons express well-characterized neuron-specific markers which include neurofilament proteins, MAP2, and class III xcex2-tubulin. Included as neurons are, for example, hippocampal, cortical, midbrain dopaminergic, motor, sensory, sympathetic, septal cholinergic, and cerebellar neurons.
By a xe2x80x9cdosage sufficient to decrease necrosisxe2x80x9d is meant an amount of a chemical compound or small molecule which when administered to a subject will decrease necrosis. Preferably necrosis is decreased in the subject 10% relative to an untreated subject. More preferably necrosis is decreased in the subject 50% relative to an untreated subject. Most preferably necrosis is decreased in the subject 90% relative to an untreated subject.
As used herein, by xe2x80x9cmeasuring necrosisxe2x80x9d is meant determining if a cell is dying through necrosis, in the presence of a compound, compared to a cell which is not in the presence of the compound (control cell). Necrosis can be measured by determining cellular ATP levels, wherein a cell that is undergoing necrosis has a decreased level of cellular ATP compared to a control cell. Necrosis may also be measured by staining with a vital dye, for example, trypan blue, wherein a cell which is necrosing will be stained with the vital dye, and a cell which is not necrosing will not be stained with the dye.
By a xe2x80x9cderivativexe2x80x9d is meant a structural derivative having a chemical modification of the compound which does not reduce the ultimate level of necrosis, but which does enhance bioavailability, solubility, or stability in vivo or ex vivo or which reduces the toxicity or dosage required. Such modifications are known to those skilled in the field of medicinal chemistry.
The present invention provides a number of advantages. For example, the methods described herein allow for a decrease in cell death occurring through a necrosis pathway. The invention also provides compounds and methods for treating diseases in which necrosis occurs. These compounds and methods can be used to treat conditions such as a neurodegenerative disease, stroke, liver disease, pancreatic disease, ischemic heart or brain injury or other ischemic injuries, head trauma, septic shock, coronary heart disease, gastrointestinal disease, tuberculosis, alteration of blood vessels, viral infection, such as HIV or AIDS, or conditions associated with a viral infection such as HIV or AIDS.
Other features and advantages of the invention will be apparent from the following detailed description and from the claims.